Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan

Abstract

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and or1B) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, oriC and orlD, which affect cell wall j0-1,3-glucan content were also identified.Walls from strains carrying mutations in these genes exhibit normal amounts of a-1,3-glucan and chitin but reduced amounts of 03-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and I0-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.The cell wall plays an important role in the growth and development of the fungi. In addition to its function as the primary osmotic barrier of the cell, the temporal and spatial regulation of wall polymer synthesis is critical to the morphogenesis of the cell types characteristic of many fungi. The architecture and synthesis of the cell walls of fungi are poorly understood. For a majority of the fungi, including most of those of medical and economic importance, chitin and/or P-1,3-glucans are believed to be the most important structural polysaccharides (3). Many studies of wall synthesis have concerned the enzymology of chitin synthase and, to a lesser extent, P-1,3-glucan synthase.In the yeasts or yeastlike fungi such as Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe, chitin is a relatively small fraction of the cell wall. In S. cerevisiae, chitin is found predominantly in the primary septa between cells (9, 10, 26). Three related genes (CHSJ, CHS2, and CALI) involved in chitin synthesis from S. cerevisiae (7,29,32) and one from C. albicans (1) have been sequenced. The finding that the CHSJ (chitin synthase 1) gene from S. cerevisiae, which codes for the major chitin synthase activity, could be disrupted without affecting chitin synthesis was unexpected (7). In addition, at least under some conditions, both the CHSJ and CHS2 genes can be disrupted without affecting the majority of chitin synthesis (5). Recently, a gene (CALl) with regions of homology to chitin synthases 1 and 2 and required for chitin synthase 3 activity was identified (32). Chitin synthase 3 was shown to be responsible for chitin synthesis in the portion of the wall not associated with the septum and to be responsible for the chitin ring formation which prec...

Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal.

“…Others affect the cytoskeleton. Examples of mutations that affect wall components include orlA, orlB, tsE, and bimG, which inhibit chitin synthesis (Shaw et al, 1991;Borgia, 1992;Borgia and Dodge, 1992), and orMC, orlD, and manA, which affect glucan and mannan synthesis (Borgia and Dodge, 1992;Markham and Bainbridge, 1992;Smith and Payton, 1994). All of these cause spherical cell swelling and lysis.…”

“…The Cell Wall of the AnudC Mutant Is Abnormal Some A. nidulans mutations that affect the synthesis of the chitin precursor GlcNAc cause morphological abnormalities similar to those caused by deletion of nudC but can be remediated by the addition of GlcNAc to the medium (Borgia and Dodge, 1992 (Figure 7). AnudC cells exhibited increased Calcofluor staining of the germling wall at swollen regions of the wall at lower temperatures and over the whole surface of spheres formed at the higher temperature.…”

“…Cell wall analysis was performed as previously described by Borgia (1992) and Borgia and Dodge (1992). Briefly, wild-type and AnudC strains were grown at 37°C for 8 h in liquid medium with agitation and cells were then collected by centrifugation.…”

Compounds active against cell walls of medically important fungi

“…It is known that the papulacandins and nikkomycins depress but do not completely halt the synthesis of beta-(1,3)-glucan and chitin, respectively (106,183). However, in studies described earlier in this review (22,23,133), temperature-sensitive mutants of A. nidulans that were completely blocked for either chitin or beta-(1,3)-glucan synthesis showed the same patterns of osmotically sensitive cells found when combinations of cilofungin and nikkomycin Z were used against A. fiumigatus (185).…”

“…When an intermediate tonicity was used, swollen hyphae were produced. More recently, two reports have described a series of temperature-sensitive mutants of A. nidulans defective either in amidotransferase (hence, blocked for chitin synthesis) or in the ability to synthesize beta-(1,3)-glucan (22,23). At the restrictive temperature, germinating conidia of the mutants deficient in chitin formed osmotically sensitive cells that lysed under hypotonic conditions; the phenotype was reversed by supplying exogenous N-acetylglucosamine.…”